DNA rearrangements play a major role in producing genetic diversity during evolution. Stichotrichous dilates rely on DNA rearrangements for the crucial process of differentiation. These tiliates have two types of nuclei, a micronudeus (MIC)and macronudeus (MAC). Duringmating and differentiation, the germline MICundergoes massive global deletions and rearrangements to form the somatic MAC. In Oxytricha trifallax, this process reduces the 1GbMICgenome (containinggenieregions and "junk" DNA) to a 50Mb MAC genome that is mostly coding DNA. The segments of DNA removed during this developmental process are called internally exdsed sequences (IBS). IBSoften interrupt genieregions (coding sequences) in the MIC and must be removed to expose the correct MAC-destined sequence (MDS). In addition to presence of IBS,the MDS fragmentsin the MIC are often disordered, or scrambled, relative to their order in the MAC. Therefore,genome rearrangements are needed to correctly sort the MDS. This proposal will investigate the mechanism of programmed DNA rearrangements in Oxytricha via experiments that will test 4 hypotheses, each addressing major gaps in current knowledge. Hypothesis 1: Conventional (non-scrambled) lESs are removed via a mechanism similar to that used by DDB transposases. Preliminary results show that a conventional IBScan be circularized upon removal. Experiments will isolate reaction intermediates to help infer the mechanistic pathway of conventional IBS removal. Hypothesis 2: Conventional and non-conventional IBSs are removed via the same mechanism. The test of this hypothesis will be the experimental conversion of a non-conventionalIBSto a conventional IBSthrough microinjection of different templates that switch the identity of a targeted IBS. Hypothesis 3: Conventional IBSs are removed before unscrambling occurs. Previous attempts to address this question in Oxytricha were incondusive. qPCR will permit monitoring of IES:MDS and MDS:MDS junctions in DNA from various points during macronudear development. Hypothesis 4: Pointer sequences (regions of microhomology) are not required for MDSunscrambling or IBS removal. Though pointers have been considered essential in this process, recent evidence suggests that maternal RNA "templates" guide gene unscrambling. Microinjectionof templates lacking pointers or bearing synthetic pointers will test this hypothesis. Health relevance: DNA rearrangements (deletions, inversions, and duplications) are a major factor contributing to genome instability assodated withmany human diseases, induding cancer, with translocations and gross deletionsresponsible for a significant portionof cancer and inherited diseases. Recombinationbetween "hotspots" can result in either deletion of tumor-suppressing genes or duplication, and subsequent over-expression, of genes that promote tumor stability. Oxytricha provides an excellent model system to study DNA rearrangement, because of the magnitude of its rearrangements and the sponsor's ability to reprogram DNA rearrangements in vivo. This makes Oxytricha unparalleled in its ability to shed light on this complex process and similar mechanism(s)responsible for cancerous genomeinstability in humans.